I intend to investigate the mechanism and regulation of transcription which is coupled to DNA replication. Coupling of these two processes occurs especially in T-even phage development but similar mechanisms may exist in some eucaryotic viruses and in cell cycle regulation. My experimental approach will focus on T4-infected E. coli cells since this model system is genetically, physiologically and biochemically most accessible. Two main aspects will be considered in elucidating the coupling phenomenon: a) What are the protein components of the transcription machinery and how do they function? b) What is the "competent" structure of the DNA template allowing the transcription of T4 late genes, how is it created and how is it recognized and used by the transcription machinery? Experiments concerning the first aspect will involve a qualitative and quantitative analysis of the interactions between host and T4-specific subunits of RNA polymerase and certain replication proteins. The functions of these proteins will be characterized by in vivo and in vitro experiments. In particular, I want to test the hypothesis that the ratio of transcription of the different T4 gene classes is determined by the chemical modification of the host RNA polymerase alpha subunit and the amount of one T4-specific protein which antagonizes the functions of the host sigma subunit. Experiments relevant to the "competent" DNA structure will include genetic, physiological and biochemical approaches to the identification and role of replication proteins involved in the creation of that structure. The selection or construction of a T4 DNA restriction fragment allowing a precise chemical and physico-chemical characterization of "competence" is intended. The experiences gathered during the proposed experiments together with information from other laboratories should allow the development of a highly defined in vitro system in which the complex mechanism of replication-coupled transcription is accessible to efficient and in-depth investigation.